Therapeutic Effect And Mechanism Of Betaine On Pulmonary Arterial Hypertension Based On PERK-eIF2α Signaling Pathway

Objective: Pulmonary arterial hypertension(PAH),arising from different etiology,is a group of diseases or clinical syndromes characterized by increasing pulmonary artery pressure and pulmonary vascular circulation resistance.PAH presents a high morbidity and mortality,which will lead to right heart failure and increase in volume load until death eventually.The abnormal proliferation and apoptosis resistance of pulmonary arterial smooth muscle cells(PASMCs)and pulmonary vascular remodeling caused by increasing pulmonary arterial pressure are considered to be the main features of the pathophysiological changes of PAH.Betaine,a quaternary amine-type water-soluble alkaloid widely distributed in animals and plants,is one of the active ingredients isolated from Lycium barbarum L.Betaine possesses a variety of pharmacological actions such as anti-inflammatory,anti-proliferative and cardioprotective effects.This study aims to further explore the therapeutic effect of betaine on PAH and its possible mechanism by constructing a rat model of PAH induced by monocrotaline(MCT)and a human pulmonary artery smooth muscle cells(HPASMCs)proliferation model induced by platelet-derived growth factor(PDGF-BB).Methods:(1)There were two models in this experiment.Sprague-Dawle rats were injected with a single subcutaneous injection of MCT(50 mg/kg)to establish rat PAH model and platelet-derived growth factor(PDGF-BB)(20 ng/ml)was used to establish HPASMCs proliferation model.(2)Echocardiography was used to detect echocardiographic parameters(PATT,PVmax and PA)in rats.(3)Right cardiac catheterization was used to detect hemodynamic parameters(mPAP,RVSP)in rats.(4)Organ weighing method was used to detect right ventricular hypertrophy(RVHI)in rats.(5)H.E.staining was used to observe and evaluate the histopathological changes of pulmonary arterioles in rats.(6)Transmission electron microscope was used to observe and evaluate the histopathological changes of ultrastructure of PASMCs and endoplasmic reticulum in rats.(7)Immunohistochemical staining was used to evaluate the expression level of GPR78 and CHOP in lung tissues of rats.(8)Western blot was used to evaluate the protein expression level of GPR78,CHOP,Caspase-12 and p-PERK in lung tissues of rats.(9)CCK-8 was used to detect and evaluate the effects of betaine on cell activity and proliferation in HPASMCs.(10)After PERK was intervened with siRNA,HPASMCs were treated with betaine.The changes of protein expression level of PERK,p-PERK,eIF2α and p-eIF2α in HPASMCs were detected by western blot.Results:(1)Echocardiography results showed that compared with the control group,PATT and PVmax were significantly decreased in rats of the MCT group,while PA was significantly increased.After the treatment with betaine(200 mg/kg and 400 mg kg),PATT and PVmax were significantly increased in rats,and PA was significantly decreased.(2)The results of hemodynamics and organ weighing method showed that the hemodynamic parameters(mPAP and RVSP)and RVHI in rats of the MCT group were significantly higher than those in the control group.After the treatment with betaine(200mg/kg and 400 mg/kg),mPAP,RVSP and RVHI in rats were significantly decreased.(3)H.E.staining results showed that the thickness and area percentage of pulmonary arterioles(WT% and WA%)in rats of the MCT group were significantly higher than those in the control group.After the treatment with betaine(200 mg/kg and 400 mg/kg),WT% and WA% of pulmonary arterioles in rats were significantly decreased.(4)Transmission electron microscopy results showed that compared with the control group,PASMCs were proliferated and enlarged in rats of the MCT group,and a large number of swollen endoplasmic reticulum could be seen in their cytoplasm.After the treatment with betaine(200 mg/kg and 400 mg/kg),PASMCs in rats proliferation were significantly reduced,and endoplasmic reticulum swelling and proliferation pathological changes were significantly improved.(5)The results of western blot and immunohistochemistry showed that the expression of GRP78 and CHOP were significantly decreased in the lung tissues of rats after the treatment with betaine(400 mg/kg).The results of western blot showed that the protein expression level of Caspase-12 was significantly decreased and the protein expression level of p-PERK was significantly increased in lung tissues of rats after the treatment with betaine(400 mg/kg).(6)The results of CCK-8 showed that compared with control group,betaine had no toxicity to HPASMCs below the concentration of 250 μM.And betaine had a significant inhibitory effect on the proliferation of HPASMCs starting from a concentration of 100 μM.(7)The results of western blot showed that betaine(100 μM)significantly upregulated PERK phosphorylation and eIF2α phosphorylation in HPASMCs.After PERK was intervened with siRNA,mRNA and protein levels of PERK were significantly decreased,suggesting that PERK siRNA significantly knocked down PERK expression levels.The results of western blot also showed that when PERK was intervened with siRNA,eIF2αphosphorylation level was significantly decreased at the same time,and betaine could restore the reduction of PERK and eIF2α phosphorylation level caused by PERK siRNA.Conclusion:1.Betaine had a therapeutic effect on MCT-induced pulmonary arterial hypertension in rats.2.Betaine inhibited the abnormal proliferation of HPASMCs induced by PDGF-BB.3.Betaine played a therapeutic role in treating pulmonary arterial hypertension by enhancing PERK-eIF2α signaling pathway,inhibiting endoplasmic reticulum stress and its target was PERK.